The isolation of pharmaceutical proteins in the crystalline state is important because crystals can be dried easily and subsequently stored at low temperature under conditions where the stability of the bulk protein is optimal. Insulins and insulin analogues in which the .epsilon.-amino group of a lysine residue contained therein has a lipophilic substituent e.g. in the form of an acyl group have a protracted profile of action and show promise for use in long-acting basal therapy in the treatment of IDDM (insulin-demanding diabetes mellitus) and NIDDM (non-insulin-demanding diabetes mellitus). The preparation of such acylated insulins and insulin analogues is described in Japanese patent application 1-254,699 (Kodama), in WO 95/07931 (Novo Nordisk), in EP 0 712 862 A2 (Eli Lilly) and in WO 96/29344 (Novo Nordisk). Unfortunately, such acylated insulins and insulin analogues have been found to be less prone to crystallize than the unmodified parent compounds.
Proinsulins, insulins and insulin analogues are labile proteins and their stability depends i.a. on the purity of the particular preparation. Optimal stability can be expected when a pure preparation is kept in solid form, in particular in the form of crystals.
Unfortunately, precipitation of these compounds from solutions usually leads to amorphous precipitates which are difficult or impossible to isolate by filtration. In stead they can be isolated by centrifugation. However, when they are isolated by centrifugation it is difficult to free the particles efficiently from the mother liquor. Even when amorphous particles can be isolated by filtration, the amorphous material will usually be less pure than corresponding crystals because more impurities are embedded in amorphous material than in crystals.
The preparation of of N.sup..epsilon.B29 -(myristoyl)des(B30) human insulin is described in European patent application No. 94926816.3, Example 33. These zinc-free crystals were precipitated at pH 9 from 20% aqueous ethanol containing 0.625M sodium chloride. A method for recovering acylated proteins, especially certain fatty acid-acylated insulins, by precipitation and filtration as a freely-flowing powder is described in EP 0 747 391 A2 (Eli Lilly). According to this method a filterable precipitate of a protein is obtained by adjusting the pH of an aqueous solution containing the protein and adding a suitable amount of alcohol. For use in therapy, proteins must be prepared in highly purified form and the storage conditions must ensure that degradation during the storage is minimized. An acylated insulin or an acylated insulin analogue in highly purified form is administered in the form of a solution of a zinc complex thereof in a composition which further comprises a phenolic compound. Accordingly, it would be convenient in the production to store the bulk acylated insulin or acylated insulin analogue in the form of filterable crystals containing both insulin or insulin analogue, zinc and a phenolic compound.
It is thus an object of the present invention to provide a method by which zinc complexes of an acylated insulin or an acylated insulin analogue, optionally also containing a phenolic compound, can be obtained in filterable, crystalline form.
According to the invention this object has been accomplished by precipitating the acylated insulin or acylated insulin analogue from an aqueous buffer.